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The Journal of Immunology, 1998, 161: 1292-1298.
Copyright © 1998 by The American Association of Immunologists

Indirect T Cell Allorecognition and Alloantibody-Mediated Rejection of MHC Class I-Disparate Heart Grafts1

Gavin J. Pettigrew, Emma Lovegrove2, J. Andrew Bradley2, John Maclean and Eleanor M. Bolton2,3

Department of Surgery, University of Glasgow, Scotland, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent studies in the rat have identified a role for T cell-dependent alloantibody in rejection of MHC class I-disparate allografts. RT1Aa-disparate PVG.R8 heart grafts are rejected acutely in naive, and hyperacutely in sensitized, PVG.RT1u recipients by CD4 T cell-dependent alloantibody. Here, we examined the T cell Ag recognition pathways responsible and show that direct injection into skeletal muscle of plasmid DNA, encoding a water-soluble form of the RT1Aa MHC class I heavy chain (pcmu-tAa), stimulates IgG2b cytotoxic alloantibody and markedly accelerates rejection of PVG.R8 heart grafts (median survival time 2 days). pcmu-tAa injection did not induce CTL to Aa, arguing against direct allorecognition of soluble Aa. Treatment with mAbs confirmed that the alloimmune response to pcmu-tAa injection depended on CD4, not CD8, T cells. Priming T cells for indirect allorecognition by injection of 15-mer peptides spanning the {alpha}1 and {alpha}2 domains of Aa failed to stimulate anti-Aa Ab but caused an accelerated Ab response to a PVG.R8 heart and a modest acceleration in graft rejection (median survival time 4 days). These results suggest that both soluble MHC class I and allopeptides prime CD4 T cells by the indirect pathway, but that soluble class I is a more effective immunogen for humoral alloimmunity because its tertiary protein structure provides B cell epitopes. We propose that priming humoral alloimmunity, like CTL priming, requires recognition of intact MHC on donor cells, but essential T cell help can be provided by CD4 T cells recognizing allogeneic class I exclusively by the indirect pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Major histocompatibility complex (MHC) class I-incompatible allografts stimulate a complex T cell-dependent immune response in which both CD8 and CD4 T cells may participate (reviewed in Refs. 1, 2). Because expression of CD4 generally defines T cell specificity for MHC class II (3, 4), the contribution of such cells to rejection of MHC class I-disparate grafts is usually attributed to recognition of allogeneic class I, not as intact protein but after it has been processed and presented as linear peptide in the context of class II MHC on the surface of recipient APC (5, 6, 7), so-called indirect allorecognition (reviewed in Refs. 8, 9). Increasing evidence from both rodent models (6, 10, 11, 12, 13) and from human studies (14, 15) suggests that T cells activated through indirect allorecognition may play an important role in the graft rejection response.

If CD4 T cells recognize class I MHC alloantigen by the indirect pathway, this raises interesting questions about the nature of the effector mechanisms by which such cells destroy donor cells bearing intact allogeneic class I target molecules. These have not been well characterized, but cellular pathways have been suggested (11, 16) whereby CD4 T cells recognizing alloantigen by the indirect pathway could, at least in principal, contribute to allograft rejection by each of the major effector mechanisms thought to be capable of destroying an allograft, namely, through release of proinflammatory cytokines resulting in delayed-type hypersensitivity and by provision of T cell help for the generation of either class I-restricted CD8 CTL or the production of alloantibody by B cells.

Recent studies in the rat have highlighted the potential role of T cell-dependent alloantibody responses in the rejection of certain MHC class I-disparate allografts. In particular, rejection of MHC class I Aa-disparate PVG.R8 grafts by PVG.RT1u congenic recipients was shown, by both adoptive transfer analysis and in vivo T cell depletion studies, to depend on the presence of alloreactive CD4 T cells, and passive transfer of immune serum demonstrated that, at least in this MHC class I-disparate rat strain combination, CD4 T cells initiate graft rejection by providing T cell help for alloantibody-dependent effector mechanisms (5, 17, 18). In contrast, CD8 T cells were found, in these studies, to be neither necessary nor, by themselves, sufficient to cause rejection of class I-disparate grafts. (5, 17, 18).

We have previously argued that CD4 T cells, in this experimental model, most likely recognize and respond to MHC class I alloantigen by the indirect pathway (17). However, the alternative possibility that CD4 T helper cells, in these experiments, are responding to additional (undefined) alloantigens expressed by donor APC or are recognizing Aa alloantigen directly on donor APC cannot be discounted (17). Clarification of this issue is of obvious importance for understanding the T cell recognition pathways underlying rejection of class I-disparate allografts. It also has a bearing on the nature of T-B cell collaboration during alloantibody production to MHC class I, since the allorecognition pathway of CD4 T helper cells (indirect or direct) dictates whether T cell help for B cells is provided by cognate (Ag-specific) or noncognate (Ag-nonspecific) cell-cell collaboration (19, 20).

In this paper, we report that immunization of PVG.RT1u rats with soluble class I MHC alloantigen, by direct injection into skeletal muscle of DNA encoding a truncated form of the rat Aa class I heavy chain, stimulates a strong CD4 T cell-dependent anti-MHC class I Ab response and causes markedly accelerated rejection of MHC class I-disparate heart grafts. In contrast, immunization with synthetic 15-mer peptides, corresponding to the hypervariable regions of MHC class I, failed to stimulate anti-class I alloantibodies before heart transplantation but led to an accelerated Ab response following transplantation and a modest acceleration in graft rejection. These results highlight the importance of the indirect pathway of allorecognition in the rejection of MHC class I-disparate grafts, particularly where Ab-dependent effector mechanisms are involved. We propose that both soluble MHC class I and synthetic allopeptides are able to effectively prime CD4 T helper cells by the indirect pathway but that soluble MHC class I is a more effective immunogen than linear allopeptides for stimulating humoral alloimmunity because its tertiary protein structure provides the conformational B cell epitopes necessary for generation of pretransplant Abs directed against target cells expressing intact allogeneic MHC class I.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Congenic PVG.RT1u (AuB/DuCu), recombinant PVG.R8 (Aav1B/DuCu), PVG (RT1c), and DA (RT1av1) rats were purchased from Harlan U.K. (Bicester, Oxon, U.K.). The derivation of the PVG.RT1u and PVG.R8 rat strains is cross-referenced elsewhere (5). All animals were maintained under standard conditions and used when 8 to 12 wk old.

DNA constructs

cDNAs encoding the full-length and a truncated, soluble form of the rat RT1Aa class I MHC molecule, in the pcexv-1-neo plasmid, were kindly provided by Dr. Simon Powis (Wellcome Trust Building, University of Dundee, U.K.). The cDNA insert in this vector is under the control of an SV40 early-late promoter, and the soluble form of the MHC class I Aa molecule differs from the original full-length sequence by the inclusion of a stop codon after the methionine residue at position 284 in the transmembrane region. In preliminary studies, plasmids encoding full-length or truncated Aa were used to transfect the rat myoblast cell line, L6 (Ref. 21, ECACC, Salisbury, U.K.) by liposome-mediated transfer using DOTAP (Boehringer Mannheim, Mannheim, Germany). Flow cytometric analysis of stably transfected L6 cells confirmed that only the full-length, and not the truncated, Aa molecule was expressed on the cell surface (Fig. 1Go). Conversely, soluble Aa, detected by ELISA using MN4-91-6 (anti-RT1Aa, 22 as the capture Ab and biotin-conjugated OX18 (anti-RT1A, 23 for detection, was present in the supernatant of cells transfected with the truncated, but not the full-length, Aa molecule.



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  		FIGURE 1. Cell-surface expression of Aa MHC class I by the rat myoblast cell line L6 following liposome-mediated transfection with pcexv-1-neo plasmid. Cells were transfected with empty plasmid or plasmid containing the DNA sequence encoding either the full-length (membrane-bound) or a truncated (water-soluble) form of the RT1Aa molecule. Flow cytometric analysis was performed after incubation of cells with FITC-conjugated MN4-91-6 mAb, which labels a polymorphic determinant of RT1Aa.

 
The eukaryotic expression plasmid, pcmu-IV (24), containing the full-length RT1Aa cDNA (25), was generously provided by Dr. Etienne Joly (Babraham Institute, Cambridge, U.K.). The RT1Aa sequence had been inserted into the pcmu-IV plasmid at the NotI and XbaI restriction sites (Fig. 2Go). For the DNA vaccination studies described in this study, an additional plasmid was constructed by excising the full-length RT1Aa sequence from the pcmu-IV plasmid and substituting it with the truncated RT1Aa cDNA (from the pcmu-1-neo plasmid) at the NotI and XbaI restriction sites. Empty plasmid (pcmu-IV) for control studies was created by excising the class I MHC insert at the BamHI sites and re-ligating the ends of the parent plasmid. Plasmid DNA was purified using a Qiagen Plasmid Megakit (Qiagen, Crawley, U.K.) incorporating Endotoxin Removal Buffer, according to manufacturer’s instructions. Typical endotoxin levels in DNA purified using this procedure are <50 endotoxin units per mg DNA (26).



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  		FIGURE 2. Plasmid map of the pcmu-IV expression vector encoding the RT1Aa {alpha}-chain. The DNA sequences for the full-length or truncated Aa {alpha}-chain were inserted at the NotI and XbaI restriction sites. The first 14 amino acids of the signal peptide, upstream of the NotI site, are provided by the corresponding region of the mouse class I (H2-Db) sequence. The truncated Aa sequence encodes a soluble form of the class I {alpha}-chain resulting from the inclusion of a stop codon at nucleotide 934 in the transmembrane region.

 
In vivo injection of plasmid DNA

Gene transfer of DNA encoding the truncated RT1Aa molecule into adult PVG.RT1u rats was achieved by direct injection of plasmid DNA into skeletal muscle (27). To induce regeneration of skeletal muscle fibers and thereby increase the efficiency of gene transfer (28, 29), 400 µl of 0.5% Bupivacaine (1-butyl-N[2, 6-dimethyl phenyl] 2-piperidine-carboxamide) were first injected into each tibialis anterior muscle using a 28-gauge needle. Three and eight days later, 200 µg of pcmu-IV encoding the truncated Aa molecule in 400 µl of saline were injected into each tibialis anterior muscle.

Monoclonal Abs

The following mouse mAbs were used for in vivo treatment: MRC OX8 (CD8, 30 and MRC OX38 (CD4, 31 . Hybridoma cells secreting these Abs were injected i.p. into pristane-primed BALB/c mice to produce ascites, from which IgG was purified by protein A column chromatography (ProSep, Fisher Scientific, Loughborough, U.K.). The OX8 and OX38 mAb treatment regimens used to induce blockade of CD8 and CD4 T cell subsets, respectively, were based on our experience with these Abs in previous studies (5, 18). The mouse IgG2a mAb, ESH8, which is directed against human factor VIII (Scottish Antibody Production Unit, Law, Scotland, U.K.), was used as an isotype control Ab (32).

Allopeptides and allopeptide immunization

A series of 18 overlapping (by 5 amino acids) 15-mer peptides that span the {alpha}1 and {alpha}2 domains of the RT1Aa molecule (residue 28 (glycine) to 212 (phenylalanine) inclusive; 25 were obtained from Immune Systems (Paignton, U.K.). The allopeptides were synthesized by standard F-moc chemistry, purified by HPLC, and assessed by mass spectrometry (peptide purity >80%).

PVG.RT1u rats were immunized s.c. in each hind footpad with a single injection of 900 µg of allopeptide (comprising a mixture of 50 µg of each of the 18 individual allopeptides), dissolved in 50 µl of water and emulsified with a comparable volume of CFA (Sigma).

Cardiac transplantation

Heterotopic cardiac transplantation was performed by the modified technique of Ono and Lindsey (33), using standard microsurgical techniques with end-to-side anastomosis of the donor aorta and pulmonary artery to the recipient infrarenal aorta and vena cava, respectively. Cold ischemic times were less than 30 min. Grafts were assessed by daily palpation, and rejection was defined as the complete cessation of myocardial contraction. Differences in graft survival were assessed by the Mann-Whitney U test. P values (two-tailed) of <0.05 were considered significant.

Skin transplantation

Recipients were grafted on the flank with full thickness skin grafts, as described elsewhere (34).

Cytotoxic alloantibody determination

Lymphocytotoxic Abs in serum samples were detected by their ability to lyse 51Cr-labeled, Con A-transformed splenic blasts in the presence of guinea pig complement, as described elsewhere (35). Percent specific 51Cr release was calculated by the formula: (experimental release - spontaneous release)/(maximum release - spontaneous release) x 100.

Class and subclass determination of anti-AaAb

The Ig class and isotype of serum Ab against RT1Aa were determined by flow cytometric analysis, using PVG.R8 pooled cervical and mesenteric lymph node cell (LNC)4 targets and FITC-conjugated mouse anti-rat Ig-specific mAbs, as described previously (35).

Cell-mediated cytotoxicity assays

LNC and spleen cells from allograft recipients were prepared and tested for their ability to lyse donor-strain, 51Cr-labeled, Con A-transformed splenic blasts in a standard 6-h 51Cr-release assay (36). Percent specific 51Cr release was calculated by the formula: (experimental release - spontaneous release)/(maximum release - spontaneous release) x 100.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of priming with an Aa-disparate skin graft or Aa allopeptides on heart graft rejection

Congenic PVG.RT1u rats respond strongly to RT1Aa class I MHC alloantigen and, as shown in Table IGo (group 1), naive PVG.RT1u animals reject Aa-disparate PVG.R8 heart grafts rapidly (MST 7 days). Prior exposure to Aa Ag on donor cells is particularly effective at inducing accelerated heart graft rejection in this class I-disparate strain combination, and PVG.RT1u recipients that were sensitized by the application of a full thickness PVG.R8 skin graft, 12 days before heart transplantation, consistently rejected PVG.R8 heart grafts within 1 day of transplantation (Table IGo, group 2). Sera obtained at the time of heart grafting, from recipients sensitized by skin grafting, showed high levels of circulating cytotoxic anti-Aa alloantibody when assayed against PVG.R8 lymphoblast targets (Fig. 3Go), and we have shown previously, by passive transfer of immune sera from sensitized into naive recipients, that such Ab are able to effect accelerated rejection of PVG.R8 heart grafts (37).


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  		Table I. Rejection of Aa class I MHC-disparate PVG.R8 heart grafts by PVG.RT1u recipients

 


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  		FIGURE 3. Circulating lymphocytotoxic RT1Aa Ab response of PVG.RT1u rats 12 days after application of a full-thickness PVG.R8 skin graft or immunization with Aa allopeptide. Allopeptide-immunized animals received 900 µg of pooled Aa peptides emulsified in CFA and given s.c. into each hind footpad. Control animals received adjuvant alone. Sera were assayed against 51Cr-labeled PVG. R8 Con A blasts in the presence of guinea-pig complement. Values shown are mean and SD of four animals per group.

 
Compared with skin grafting, immunization of PVG.RT1u rats with synthetic allopeptides corresponding to the hypervariable regions of the Aa class I MHC molecule is less effective at promoting accelerated rejection of Aa heart grafts (Table IGo). In a recent study (37) we showed that PVG.RT1u rats immunized s.c., with an emulsion of CFA and a single allopeptide corresponding to the {alpha}-helical region of either the {alpha}1 (amino acids 57–80) or the {alpha}2 domain (amino acids 143-163) of the Aa molecule, reject heterotopic PVG.R8 heart grafts marginally, but significantly, faster than control animals given CFA and control peptide (MST 5 vs 6 days, respectively, p < 0.02). Because these peptides did not encompass all of the potential T cell epitopes in the {alpha}1 and {alpha}2 domains of Aa. this may have limited the efficacy of sensitization. For this reason, those experiments were extended here by immunizing PVG.RT1u rats with a mixture of 18 overlapping 15-mer peptides spanning the entire {alpha}1 and {alpha}2 domains (amino acids 28–185, inclusive). Pooled allopeptides emulsified in CFA were injected s.c. into the hind footpads of recipient animals 12 days before they received a PVG.R8 heart allograft. As shown in Table IGo (groups 3 and 4), recipients immunized with allopeptide rejected their heart grafts faster than control animals given CFA alone (MST 4 days vs 6.5 days, respectively, p < 0.02), but heart graft rejection in allopeptide-primed animals was still markedly slower than that observed in animals primed by a PVG.R8 skin graft (p < 0.02). The kinetics of heart graft rejection in animals immunized with CFA alone in this study were comparable to those observed in our previous study (37), when animals were immunized with CFA emulsified with an irrelevant control peptide (MST 6.5 and 6.0 days, respectively).

In this, as in our earlier study (37), immunization with Aa allopeptides did not stimulate the development of cytotoxic Ab recognizing intact RT1Aa on target cells, and sera obtained from peptide-primed animals on the day of heart grafting, i.e., 12 days after immunization, showed only background levels of PVG.R8 target cell lysis (Fig. 3Go). However, by day 4 after heart transplantation, recipients that had been immunized with allopeptides showed higher serum levels of cytotoxic alloantibodies than control animals immunized with CFA alone before heart transplantation (Fig. 4Go). Together, these observations are consistent with the idea that immunization with linear allopeptides is able to prime, via the indirect pathway, CD4 T cells that provide B cell help, but does not provide the relevant Aa conformational B cell epitopes for anti-Aa Ab production.



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  		FIGURE 4. Cytotoxic RT1Aa Ab response of PVG. RT1u rats 4 days after receiving a heterotopic PVG.R8 heart transplant. Recipients were preimmunized with pooled Aa allopeptides emulsified in CFA (as detailed in the legend to Fig. 2Go), or with CFA alone, 12 days before heart transplantation. Results shown are mean and SD of four animals per group.

 
DNA transfer studies

To define further the nature of Aa class I MHC alloantigen necessary to initiate alloantibody production and accelerated rejection of Aa-disparate heart grafts, PVG.RT1u rats were immunized by direct injection into skeletal muscle of pcmu-IV plasmid encoding a truncated form of the Aa class I MHC molecule (pcmu-tAa). Because the truncated Aa protein lacks cytoplasmic and transmembrane regions and is not, therefore, expressed on the cell surface (see Materials and Methods and Fig. 1Go), we reasoned that immunization with pcmu-tAa would not be effective in priming alloreactive CD4 T cells by the direct pathway but that soluble Aa protein resulting from gene transfer might provide a ready source of class I MHC alloantigen for recognition by CD4 T cells through the indirect pathway as well as relevant conformational B cell epitopes for stimulating B cells with specificity for the intact Aa molecule. These predictions were supported by the observation that i.m. injection of pcmu-tAa on two occasions before heart transplantation was very effective at priming PVG.RT1u rats to Aa alloantigen and led to a marked acceleration in the rejection of PVG.R8, but not third-party PVG.RT1c, heart grafts (MST 2 days, p < 0.02, and 6 days, NS, respectively, Table IIGo, groups 2 and 3). Control recipients injected with "empty" pcmu-IV plasmid rejected PVG.R8 heart grafts at the same rate as naive PVG.RT1u recipients (MST 7 days, NS, Table IIGo, group 1).


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  		Table II. Accelerated rejection of R8 heart grafts by PVG.RT1u recipients after vaccination with DNA encoding water soluble Aa class I MHC

 
In contrast to allopeptide immunization, injection of rats with pcmu-tAa led to high levels of circulating cytotoxic anti-RT1Aa Ab before heart transplantation (Fig. 5Go). The class and subclass of the resulting anti-Aa Ab was characterized by flow cytometric analysis using PVG.R8 target cells and FITC-conjugated anti-Ig mAb. As shown in Figure 6Go, immunization with pcmu-tAa stimulated a strong IgG alloantibody response that was predominantly of the IgG2b subclass; in the rat, this is the most effective IgG subclass for mediating complement-dependent cell lysis (38).



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  		FIGURE 5. Cytotoxic RT1Aa alloantibody response of PVG.RT1u rats 12 days after immunization with pcmu-IV vector encoding the truncated Aa class I MHC sequence (pcmu-tAa). Control animals were immunized with empty pcmu-IV plasmid. Animals received 400 µl of 0.5% Bupivacaine i.m. followed, 3 and 8 days later, by injection of 200 µg of plasmid. Values shown are mean and SD of four animals per group. Shown also for comparison is the cytotoxic Ab response in animals sensitized 12 days earlier by a PVG.R8 skin graft (data from Fig. 2Go).

 


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  		FIGURE 6. Ig class and subclass of serum anti-RT1Aa Ab response after immunization with pcmu-tAa. PVG.RT1u rats were immunized with pcmu-tAa, as described in the legend to Figure 5Go, and 12 days later serum was assayed for Aa alloantibody by flow cytometric analysis using PVG.R8 LNC as targets and FITC-conjugated mouse anti-rat Ig subclass-specific mAb. Results (as mean channel fluorescence) are shown as mean and SD for three animals. Sera obtained from animals immunized with empty plasmid contained no detectable alloantibody (data not shown).

 
CTL responses

To determine whether immunization with DNA-encoding soluble Aa induced a CTL response, spleen cells were obtained from PVG.RT1u rats that had been immunized with pcmu-tAa or with a PVG.R8 skin graft and assayed against 51Cr-labeled PVG.R8 Con A blasts. Lymphoid cells from PVG.RT1u recipients primed with PVG.R8 allografts do not generally display very high levels of in vitro cytotoxic T cell activity (5). However, as shown in Figure 7Go, spleen cells from animals grafted with PVG.R8 skin showed significant levels of CTL activity whereas cells from animals immunized with pcmu-tAa displayed minimal cytotoxicity. In additional experiments, LNC obtained from PVG.RT1u rats 12 days after immunization with pcmu-tAa were stimulated in vitro for 5 days with irradiated PVG.R8 spleen cells, and CTL activity was then determined. After in vitro stimulation, the level of CTL activity observed in LNC from animals primed with pcmu-tAa were comparable to those seen in T cells from control animals immunized with empty pcmu-IV plasmid (Fig. 8Go).



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  		FIGURE 7. Analysis of CTL response to RT1Aa. PVG.RT1u rats were grafted with PVG.R8 skin or immunized with pcmu-tAa or with pcmu-IV (empty vector). After 12 days, spleen cells were assayed against 51Cr-labeled R8 Con A blast target cells in a 6-h cytotoxicity assay. Values shown are mean and SD of three animals per group. This experiment was repeated on two occasions with the same result.

 


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  		FIGURE 8. In vitro generation of CTL to Aa alloantigen. LNC were obtained from PVG.RT1u rats 12 days after immunization with pcmu-tAa (or pcmu-IV control plasmid) and used as responders (4 x 107 in 20 ml) in a bulk MLR. Stimulators were irradiated (20 Gy), fully allogeneic (RT1a) spleen cells (2 x 107). After 5 days of in vitro stimulation, responders (from three animals per group) were tested for their ability to lyse 51Cr-labeled PVG.R8 Con A blast targets in a 6-h cytotoxicity assay. Values shown are mean and SD. There was minimal lysis of third-party Lewis (RT1l) lymphoblast target cells.

 
In vivo T cell depletion studies

Finally, to determine whether the ability of immunization with pcmu-tAa to promote accelerated rejection of PVG.R8 heart grafts was dependent on CD4 or on CD8 T cells, recipients were treated in vivo with mAbs directed against T cell subsets following DNA injection. As shown in Table IIGo, the ability of PVG.RT1u animals injected with pcmu-tAa to reject PVG.R8 grafts very rapidly was not prevented by treatment with the anti-CD8 mAb, MRC OX8 (MST 1 day, groups 4 and 5). We have confirmed, previously, that treatment with OX8 mAb is highly effective at depleting T cells expressing CD8 from both the blood and lymphoid tissue of PVG.RT1u rats (5) and, in the present study, we showed that this phenotypic depletion was accompanied by functional depletion of CD8 CTL precursors. LNC obtained from unmodified PVG.RT1u rats and cultured in vitro for 5 days with fully allogeneic RT1Aa-irradiated splenic stimulators (as described in legend to Fig. 8Go) developed high levels of CTL activity against PVG.R8 lymphoblasts (>50% cytotoxicity at E:T ratio of 100:1), but CTL activity could not be generated from LNC obtained from PVG.RT1u animals during the first week after in vivo treatment with OX8 mAb (<10% cytotoxicity at E:T ratio of 100:1).

In contrast to anti-CD8 mAb treatment, in vivo treatment of pcmu-tAa-immunized PVG.RT1u rats with the anti-CD4 mAb MRC OX38, which produces depletion of approximately 50% of peripheral CD4 T cells (18), not only prevented accelerated heart graft rejection but extended graft survival well beyond that observed in naive PVG.RT1u recipients (MST 13 days, Table IIGo, group 6).

The effects of in vivo treatment with mAbs to T cell subsets on the cytotoxic RT1Aa Ab titer following immunization with pcmu-tAa and heart grafting are shown in Figure 9Go. It can be seen that administration of anti-CD4 mAb completely abrogated the early cytotoxic alloantibody response, whereas cytotoxic alloantibody responses in anti-CD8--treated recipients were similar to those observed in control animals.



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  		FIGURE 9. Effect of in vivo treatment with mAb to T cell subsets on cytotoxic RT1Aa Ab response following immunization with pcmu-tAa. PVG.RT1u rats were treated with anti-CD4 (5 mg OX38 (or isotype control, ESH8) given i.p. on days -3, 0, 3, 6, and 9 relative to plasmid injection) or anti-CD8 mAb (2 mg OX8 on days -1, 0, and 1). Sera (four animals per group) were assayed against 51Cr-labeled PVG.R8 Con A blasts. Results are expressed as mean and SD of Ab titer (last dilution of serum that gave >=20% cytotoxicity).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Rejection of MHC class I-disparate allografts has, historically, been attributed to CD8 cytotoxic effector cells with specificity for direct recognition of allogeneic MHC class I expressed by target cells within the graft (1, 2). However, several recent studies have shown that CD4 T cells may be sufficient to initiate rejection of class I-disparate allografts and, at least in the rat, CD8 T cells are not essential for effecting graft rejection (2, 5, 17, 18). Since CD4 T cells participating in rejection of class I-disparate grafts most likely recognize allogeneic class I MHC by the indirect pathway, the effector mechanisms by which T cells of this allospecificity destroy target cells expressing intact MHC class I have been the subject of considerable speculation. Our own recent studies of MHC class I-disparate graft rejection in the rat have identified a hitherto neglected role for T cell-dependent alloantibody-mediated effector mechanisms in graft rejection (5, 17, 18).

In the present study, the approach of in vivo gene transfer was used to provide insight into the Ag recognition pathways of allogeneic MHC class I. Direct injection of naked plasmid DNA into skeletal muscle has been shown, for a variety of protein Ags, to be an effective means for inducing a strong humoral and cellular immune response to the Ag encoded by the plasmid DNA (39, 40). We found that i.m. injection of DNA encoding a truncated, water-soluble form of rat MHC class I heavy chain resulted in the development of a strong CD4 T cell-dependent cytotoxic alloantibody response that led to accelerated rejection of class I-disparate heart allografts. In previous studies (2, 5, 18), we were not able to discount the possibility that, in the recombinant PVG.R8 and PVG.RT1u class I-disparate rat strains used, CD4 T helper cells were activated, not by MHC class I alloantigen, but by other undefined alloantigenic differences coexpressed by donor cells. The observation here, that DNA encoding only allogeneic MHC class I is sufficient to stimulate a strong CD4-dependent alloantibody response, demonstrates clearly that class I alloantigen alone is sufficient to activate alloreactive CD4 T helper cells. There is no need, therefore, to invoke additional putative alloantigens to explain the alloimmune response.

The results described here in the PVG.RT1u rat, where alloantibody is known to effect graft rejection, contrast with those obtained by Geissler et al. in the only previously published report of in vivo gene transfer with DNA-encoding soluble MHC class I (41). They found, in the Lewis rat strain (where alloantibody is not known to effect rejection), that injection of DNA-encoding soluble Aa MHC class I caused only a modest shortening of heart graft rejection (from 6 to 5 days) and did not consistently provoke an anti-class I alloantibody response (41). Whether the differences between these results and those reported here are attributable exclusively to differences in the ability of the two rat strains used (PVG.RT1u and Lewis) to mount an alloimmune response to soluble Aa, or whether they also reflect differences in the efficacy of gene transfer between the two studies, is not clear.

The pcmu-tAa plasmid construct was chosen for the experiments described here on the basis that it would provide, after in vivo injection, a ready source of secreted water-soluble MHC class I heavy chain, both for recognition by alloreactive B cells through surface Ig and for priming of T helper cells by the indirect pathway. We reasoned that pcmu-tAa injection would be unable to prime alloreactive T cells by the direct pathway of allorecognition because it would not result in the display of intact Aa protein on the surface of APCs. The in vitro transfection studies confirmed that, although truncated Aa protein was released into culture supernatant, it was not detectable, by flow cytometry, on the surface of stably transfected cells. Intact soluble MHC class I, released from transfected cells in vivo, could potentially interact directly with the TCR of alloreactive T cells, but such interaction would not be expected, in the absence of essential costimulatory signals delivered by APCs, to trigger T cell activation (42, 43, 44). The validity of these assumptions, which are central to the interpretation of the experiments described here, receives strong support from the analysis of CTL responses to soluble Aa. Following pcmu-tAa injection in vivo, there was no evidence for priming of CTL with specificity for direct recognition of Aa MHC class I on target cells.

In some experimental systems, soluble forms of MHC class I have been shown to exert potent immunoregulatory effects, interacting directly with alloreactive CD8 T cells to block CD8 cytotoxic activity and induce apoptosis (45, 46, 47). These immunoregulatory effects appear to be dependent, in large part, on the ability of multimeric forms of MHC class I to cause cross-linking of the TCR (45, 46, 47). In the present study, it is likely that soluble MHC class I resulting from gene transfer is released in monomeric form because it lacks a hydrophobic transmembrane tail. The inability of pcmu-tAa injection to down-regulate the CTL response to Aa MHC class I may be attributed, therefore, to the likelihood that soluble MHC class I, even if released in amounts sufficient to block CTL, is not present in a form that will result in effective cross-linking of TCR on CD8 T cells. Interestingly, Wang et al. (48) observed that s.c. immunization of PVG.RT1u rats with soluble Aa class I MHC heavy chain protein, produced in a baculovirus expression system, also primed for heart allograft rejection. Heart allograft rejection was not as rapid as in the present study, and the effect of Aa immunization on the humoral immune response was not determined.

In contrast to soluble MHC class I, priming with synthetic allopeptides did not, in this or in our previous experiments (37), stimulate a cytotoxic Aa alloantibody response, nor was it as effective in accelerating heart graft rejection. PVG.RT1u rats, immunized by s.c. injection of allopeptides corresponding to the {alpha}1 and {alpha}2 domains of Aa MHC class I, rejected Aa-disparate heart grafts more quickly than control animals (MST 4 vs 6.5 days), but graft rejection was less rapid than after immunization with pcmu-tAa plasmid (MST 2 days). Shirwan et al. also found, in the PVG.RT1u rat strain, that immunization with Aa allopeptides led to only a marginal decrease in survival of PVG.R8 heart allografts when compared with control animals (mean graft survival 5 vs 6 days, 49 , and these results for class I-disparate grafts are typical of those reported in other rat strain combinations, where immunization with synthetic MHC class I allopeptides has been shown to shorten the survival of fully allogeneic skin and heart grafts by, at most, 2 days (11, 50).

In contrast to immunization with allopeptides, sensitization of PVG.RT1u rats with intact MHC class I on the surface of donor APCs by application of an Aa-disparate PVG.R8 skin allograft, as in the present study, or by s.c. injection of irradiated PVG.R8 spleen cells, as in the study of Shirwan (49), led to prompt rejection of Aa-disparate heart grafts within 1 to 2 days of transplantation. Shirwan suggested that immunization with donor cells expressing intact RT1.Aa was more effective than allopeptide because it primed T effector cells recognizing RT1.Aa directly (49). The results of the present study suggest that the immunogenicity of donor cells may also reside in their ability to generate a humoral alloimmune response that is contingent on the presence of conformational B cell epitopes displayed on intact allogeneic MHC class I. Priming of humoral effector mechanisms is, therefore, analogous to priming of CD8 CTL (51), in that there is a requirement for recognition of intact allogeneic MHC class I expressed by and, in the case of Ab, derived from donor cells, but essential T cell help for the generation of these effector mechanisms can be provided by CD4 T cells recognizing allogeneic MHC class I exclusively by the indirect pathway.


   Footnotes
 
1 This work was supported by the National Kidney Research Fund, the Western Infirmary (Glasgow) Kidney Research Fund, and the British Heart Foundation. Back

2 Current address: University of Cambridge Clinical School, Department of Surgery, Box 202, Level E9, Addenbrooke’s Hospital, Cambridge CB2 2QQ, U.K. Back

3 Address correspondence and reprint requests to: Dr. E. M. Bolton, University of Cambridge Clinical School, Department of Surgery, Box 202, Level E9, Addenbrooke’s Hospital, Cambridge CB2 2QQ, U.K. E-mail address: Back

4 Abbreviations used in this paper: LNC, pooled cervical and mesenteric lymph node cells; MST, median survival time; pcmu-tAa, pcmu-IV plasmid encoding truncated (soluble) RT1Aa class I MHC. Back

Received for publication January 12, 1998. Accepted for publication April 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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